Process for producing zearalenone

ABSTRACT

Zearalenone is produced in an agitated, aerated, aqueous fermentation medium containing submergible, zearalenone-producing strains of Gibberella zeae and nutrients including assimilable carbon, nitrogen, and mineral sources.

United States Patent Keith et al.

[ 51 May 9, 1972 [54] PROCESS FOR PRODUCING ZEARALENONE [72] Inventors:Chester L. Keith, Manhattan, Kans. [73 Assignee: Commercial SolventsCorporation [22] Filed: June 18, 1970 [21] Appl. No.: 47,637

[52] U.S.Cl.....

51 Int.Cl. [58] Field of Search .195/36 R, 195/81, 424/279 ..C12d 13/00..l95/36 R, 81; 424/279 [56] References Cited UNITED STATES PATENTS [57] ABSTRACT Zearalenone is produced in an agitated, aerated, aqueousfermentation medium containing submergible, zearalenoneproducing strainsof Gibberella zeae and nutrients including assimilable carbon, nitrogen,and mineral sources.

18 Claims, No Drawings having the structural formula:

(also called F.E.S., for Fermentation Estrogenic Substance) bycultivation of the microorganism Gibberella zeae (Gordon) on a suitablenutrient is taught in US. Pat. No. 3,196,019. The production methoddisclosed in the patent involves incubating either the spores or thevegetative mycelia of the microorganism in a suitable inoculationmedium, and then introducing the resultant inoculation medium into afermentation medium containing one of the common grains, e.g., corn infinely divided form, as a source of carbohydrate. After a fermentationperiod of about 6 to 20 days, or more, the zearalenone so produced isrecovered from the fermentation medium. The use of such a process,involving, as it does, the utilization ofa solid phase fermentationmedium, requires the employment of a generally more difficult recoveryprocedure than would be the case were a liquid phase fermentation mediumused and the microorganism cultivated by submerged fermentation therein.Prior to the present invention, however, attempts to produce zearalenoneby submerged fermentation have been decidedly unsuccessful.

lt has now been surprisingly found that submergible, aerobiczearalenone-producing strains of Gibberella zeae (cultures of this typeare on unrestricted deposit at the American Type Culture CollectionATCC), will efficiently produce zearalenone when cultivated bysubmersion in an agitated, aerated, aqueous, liquid phase fermentationmedium containing nutrients including assimilable carbon, nitrogen andmineral sources. These strains are submergible, zearalenoneproducingcultures of Gibberella zeae and include Gibberella zeae (Schw.) Petchstrain 542 Keith ATCC 20273 and Gibberella zeae (Schw.) Petch strainPaul S. ATCC 20271.

Preferably, distilled water, deionized water, or tap water which hasbeen heated to boiling and then filtered is used in the medium. Aerationof the medium is preferably effected by bubbling air therethrough,preferably sterile air, and preferably at a rate of about 0.25 to 2volumes of air, calculated at atmospheric pressure, per volume ofmedium, per minute. The temperature of the medium is preferablymaintained at about 20 to 28, most preferably about 21 to 24 C. Duringthe early stages of the fermentation, for example until zearalenoneproduction has started, it is preferred not to allow the temperature toget much above 24 C. The medium is advantageously agitated to disperseand make air available to the microorganism and this can be effected byany suitable means, e.g., by a stirrer operating at about 200 to 500 RPMin a 20 liter fermenter.

The assimilable carbon source in the fermentation medium isadvantageously glucose, e.g., reagent grade glucose. Cerelose, a white,crystallized, refined glucose, is a suitable source -of glucose for theprocess. Also suitable, but less preferred, carbon sources are othercarbohydrates which will not deleteriously affect the production ofzearalenone, such as xylose, fructose, sucrose, and galactose. Xyloseperforms better when used in admixture with glucose (for example, at aratio of 7.5 parts by weight of xylose to 22.5 parts by weight ofglucose) than when used as the sole carbon source. If desired, a portionof the glucose, when employed, say up to about 50 weight percentthereof, can be substituted with glycerol. As an alternative tosupplying actual glucose to the fermentation medium, a precursorthereof, such as starch, which, under the conditions of the process,will be converted to glucose, can also be used. The amount ofassimilable carbon source used in the fermentation medium is that whichis sufficient for production of zearalenone and, for example in the caseof glucose, will generally range from about 20 to about 40, preferablyabout 25 to 35, grams of glucose per cc. of the medium.

The assimilable nitrogen source for the fermentation medium can beeither inorganic or organic, but preferably it is the latter.Ammonia-supplying, neutral compounds that are easily hydrolyzable aregenerally suitable. Examples of suitable nitrogen-supplying compoundsare urea, asparagine, ammonium salts such as ammonium nitrate andammonium fumarate. glutamine, glycine, ammonium hydroxide, andSheffields NZ Amine Type HD, a protein hydrolyzate. The preferrednitrogen source is urea, and this is generally employed in an amount inthe range of about 0.2 to 0.8, preferably about 0.3 to 0.7, grams per100 cc. of the fermentation medium.

The mineral sources included in the fermentation medium as nutrientsinclude the elements, iron, phosphorus, potassium, sulfur, andmagnesium, preferably in water soluble form. The amounts of the variousminerals can vary considerably although each should be present in anamount sufficient to insure proper growth of the microorganism.Generally effective amounts of each of these elements, calculated as thefree element, range from about 0.001 to 1 gram per 100 cc. of themedium. Preferred sources of these elements include dipotassium phospate(i.e., K HPO 4), magnesium sulfate (e.g., supplied as MgSO '7l-l O),potassium chloride, and, as a source of iron, sulfur and phosphorus, theaforementioned NZ Amine Type A. The potassium chloride can also functionin the medium as an osmotic pressure-enhancing salt, as is hereinafterdiscussed. The preferred amount of dipotassium phosphate employed isabout 0.05 to 0.3 grams per 100 cc. of medium, and the preferred amountof magnesium sulfate (calculated as MgSO,-7H O) is about 0.025 to 0.2grams per 100 cc. of medium. Iron is an especially desired ingredient ofthe medium when the carbon source is reagent grade glucose.

There is preferably included in the fermentation medium used in theprocess of the present invention yield-enhancing amounts of a proteinhydrolyzate, e.g., an enzymatic hydrolyzate or protein, most preferablyan enzymatic hydrolyzate of casein, such as Sheffield's NZ Amine Type Aand Amber's ECH. The Sheffield N-Z-Amine Type A is a pancreatichydrolyzate of casein which contains, in the form of mixed amino acidsand peptides, all amino acids originally present in casein. Thehydrolyzate is preferably present in the medium in an amount of at leastabout 0.1 grams, say about 0.1 to 2 grams, per 100 cc. of the medium.The most preferred hydrolyzate is NZ Amine Type A. Less preferred, butoperable, substitutes for the hydrolyzate are vitamin-free casein andcasein itself. The use of an enzymatic hydrolyzate, for example NZ AmineType A, has been indicated to be particularly useful where the carbonsource employed is reagent grade dextrose.

There is also preferably included in the fermentation medium used in thepresent invention an amount of yeast extract, e.g., Difco yeast extract(sometimes called Bacto yeast extract), sufficient to enhance the yieldof zearalenone. Generally suitable amounts of yeast extract range fromabout 0.05 to 0.2 grams per 100 cc. of the fermentation medium. Ananalysis of the composition of the mineral elements contained in Difcoyeast extract is described in Journal of Bacteriology, Vol. 84, p. 869(1962), hereby incorporated by reference. Other operable substitutes foryeast extract in the medium are corn gluten meal, corn steep liquor,Pharmamedia (a cotton seed-derived protein nutrient), Fermamine IV (anenzymatic digest of proteins), BYF300 (a fraction of autolyzed Brewersyeast), and Nutrisoy (a defatted edible soy flour), however, yeastextract is preferred.

Growth promoting amounts of animal amino acids, e.g., about 0.1 to 0.3grams per 100 cc. of the medium, of beef extract such as Difcos beefextract, can also be included in the medium.

indicated to be the least deleterious, so far as zearalenone yields areconcerned, are the silicone foam inhibitors, e.g., Antifoam 10 (anon-ionic silicone emulsion containing about 10 percent silicone solids,a General Electric Co. product). Others which are effective. but whichreduce zearalenone yieldssomewhat, include corn oil, lard oil, mineraloil, and fatty alcohols such as lauryl alcohol.

An osmotic pressure-enhancing salt can also be included in thefermentation medium used in the process of the present invention.Examples of such are the alkali metal salts, e.g., sodium acetate,sodium citrate, sodium succinate, sodium chloride, and potassiumchloride. Most preferred are the alkali metal halides, e.g. sodiumchloride and potassium chloride, and these are preferably present in anamount in the range of about 1 to 4 grams per 100 cc. of the medium.

Zearalenone yields and carbon source utilization appear to be improvedwhen the medium is sterilized by autoclaving, for example for about 10to 30 minutes for a volume up to 3 liters at aboutlO to 20 psig ofsteam, prior to being inoculated.

The fermentation is advantageously allowed to proceed, in the process ofthe present invention, until substantially all of the assimilable carbonis used, generally for about to 23 days, before the medium is subjectedto a recovery treatment to recover the zearalenone therefrom. Thezearalenone recovery can be efl'ected by any suitable procedure, forexample by filtering the medium, slurrying the filter cake with anaqueous alkaline solution so as to dissolve the zearalenone, filteringthe slurry, acidifying the filtrate so as to precipitate out thezearalenone, and then recovering the precipitated zearalenone, whichmethod is described in U.S. Pat. application, Ser. No. 721,604 of Hidyand Young, filed Apr. 16, 1968, and herein incorporated by reference.

The initial pH of the fermentation medium will generally be about 6.1 to7.2, preferably about 6.2 to 7.0. As the fermentation'proceeds, the pHdeclines. It will usually decline to a low of, say, about 3.4 to 4.0,often about 3.6 to 3.7, within about 2 to 4 days and will continue atthese pH levels for the remainder of the fermentation. if nothing isthen added to the medium to adjust the pH, it will remain at that levelfor the duration of the fermentation period. Fortunately, relatively fewcontaminating organisms can multiply at pH values below about 4.

The following examples are offered to illustrate the present invention.

EXAMPLE 1 Production of Gibberella zeae (Schw.) Petch Strain 542 KeithATCC 20273 Macroconidia of Gibberella zeae strain ATCC 20028 weretreated with a chemical mutagen according to the following procedure:

Macroconidia (about 1.3 X /ml.) were suspended in phosphate buffer of pH7.0 and dilutions were made from this stock to give 30-40 colonies perplate of an untreated culture on the plating medium described below.There was then added to he melted and cooled (45 C.) medium a sufficientamount. of N-methyl-N'-nitro-N-nitrosoguanidine to give a finalconcentration of 2 micrograms per ml. Aliquots of conidial dilutionswere added and the medium mixed and poured into Petri plates. The plateswere incubated at 30 C. until colonies had developed to a sufficientsize to permit visual inspection of colonial characteristics. Theplating medium employed had the following composition:

Plating Medium 15.0 grams per liter 5.0 grams per liter 5.0 grams perliter l-C stine 0.2 grams per liter Sod urn Chloride 4.0 grams-per liter1 Sodium Sulflte 0.2 grams per liter Sodium Citrate 1.0 grams per literA sr 15.0 grams per liter D stilled Water balance Clones were picked onthe basis of gross colonial morphology and pigmentation distinctive fromstrain ATCC 20028 and were maintained as agar slant cultures untiltesting.

The following is a morphological description of the organism grown onCzapeks dextrose agar. which has the composition:

Czapek's Dextrose Agar Sodium Nitrate 2.0 grams/liter Potassium Chloride0.5 grams/liter Magnesium Sulfate 0.5 grams/liter Ferrous Sulfate 0.01grams/liter Dipotassium Phosphate 1.0 grams/liter. Dextrose 50.0grams/liter ar 15.0 grams/liter D stilled Water balance normally appearred overall. The reverse is a deep red as the.

result of the production of water insoluble pigment.

Production of macroconidia does not normaily occur in darkness but isabundant under light. The macroconidia are borne in slimy masses,variable in length, ranging from 10 microns to microns, andcommonlycontain from 3 to 5 septations but range from 0 to 9 septations.Macroconidia are oxhorn shaped and frequently have a demonstrable footcell. The width of macroconidia is more constant, ranging from 2.0 to5.0 microns, generally around 4.0 microns. Neither microconidia norperithecia have been observed on laboratory media.

in the below-described sporulation medium macroconidia developabundantly, borne singly on a simple conidiophore. Macroconidia do not,however, develop in complex liquid media of the type described.Chlamydospore formation is prevalent in older cultures, the spores beingboth intercalary and terminal.

Macroconidial Sporulation Medium Yeast Extract (Difco) 1.0 gram/literPotassium Chloride 0.5 gram/liter Magnesium Sulfate 0.5 gram/literSodium Nitrate Ferrous Sulfate Ammonium Nitrate 0.15 gram/liter 0.007gram/liter 0.55 gram/liter Dipotassium Phosphate 1.0 gram/liter Dextrose5.8 gram/liter Distilled Water balance Complex Medium-Non SporulationDifco Yeast Extract Sodium Chloride Distilled Water 0.1 grams per 100cc. medium 0.25 grams per 100 cc. medium balance One hundred ml. of theabove medium was placed in a 500 ml. Erlenmeyer flask and autoclaved at15 psig for 15 minutes. Spores of Giberella zeae (Schw.) Petch strain542 Keith ATCC 20273 were then added to the medium and incubated thereinfor 24 hours at 30 C. on a rotary shaker. The resultant materi al wasused to inoculate a second 100 ml., autoclaved portion of the modifiedBennetts medium, and this second stage was also incubated for 24 hoursat 30 C. on a rotary shaker.

Fermentation Medium Cerelose 33 g. per 100 cc. medium Urea 0.4 g. per100 cc. medium Bacto Yeast Extract 0.1 g. per 100 cc. medium NZAmine-Type A (Sheffield) 0.3 g. per 100 cc. medium MgSO '7H O 0.025 g.per 100 cc. medium K,HPO 0.05 g. per 100 cc. medium KCL 0.025 g. per 100cc. medium Distilled Water balance One hundred (100) ml. of the abovemedium was placed in a 500 ml. flask and autoclaved at 15 psig steam for15 minutes. There was then added to the flask as an inoculum, cc. of thesecond stage material obtained from above. The inoculated medium wasthen incubated for 2 weeks at 20 C. on a rotary shaker turning at 300RPM, at the end of which period zearalenone was determined to be presentin the medium in a concentration of 15.2 grams per liter.

EXAMPLE Ill To 1,500 ml. of autoclaved modified Bennett's medium in a6-liter Erlenmeyer flask was added 100 ml. ofa first stage seed materialof ATCC 20273 prepared as in Example ll. The flask was then incubated ona reciprocating shaker at 30 C. for 24 hours to yield a final inoculumstage.

Fermentation Medium Difco Yeast Extract 15.0 grams NZ Amine-Type A 45.0grams KCl 3.75 grams MgSO '7H,O 3.75 grams l(,l-lPO 7.5 grams Cerelose4950 grams Urea 61.5 grams Distilled Water General Electrics Antifoam 10(to bring to 13.5 liters) 250 RPM, and the temperature of the medium wasmaintained at 20 C. After 15 days the zearalenone concentration reached12.529 grams per liter.

EXAMPLE IV Fifty (50) gallons of the modified Bennetts medium of Examplell was placed in a gallon stainless steel fermentor and thereinsterilized with 121 C. steam for 15 minutes. After cooling to about28-30 C., the medium was inoculated with 3 liters of an inoculum of ATCC20273 which had been prepared as in Example 11. The inoculated mediumwas then aerated (3 cubic feet per minute of air at 2-3 psig) andstirred (100 RPM) for 24 hours at 28-30 C., whereupon the culture growthwas judged to be sufficient (280 mg. per 100 ml. of medium) for use asinoculum in a production phase fermentatron.

Fermentation Medium Bacto Yeast Extract 265 grams KCl 66 grams M gSO,-7HO 66 grams K HPO 133 grams Cerelose 193 pounds Urea 1087 grams GeneralElectrics Antifoam 10 60 ml.

Distilled Water (to bring to 70 gallons) The above fermentation mediumwas placed in a 100 gallon fermentor, sterilized with steam for 15minutes at 121 C., cooled to 20 C., and inoculated with approximately 6liters of the seed culture. The temperature of the medium was thenmaintained at 20 C. and sterile air was bubbled through the medium at ahead pressure of 3 psig (4 CFM) while the medium was stirred at 200 RPM.After 8 days the pH of the medium was 3.8. It was then adjusted to 8.0by addition of urea and ammonium hydroxide. The pH was then brought downto 5.6 by the addition of nitric acid. On the 19th day of operation thelevel of zearalenone in the medium was 8.829 grams per liter, and thevolume of the medium was 64 gallons.

EXAMPLE V The procedure of Example III is used under essentially thesame conditions except Gibberella z'eae (Schw.) Petch strain Paul S.ATCC 20271 is used instead of ATCC 20273 to produce zearalenone.

It is claimed:

1. A process for the production of zearalenone which comprisescultivating a submergible, aerobic zearalenone-producing strain of themicroorganism Gibberella zeae while the microorganism is submerged in anagitated, aerated, aqueous, liquid phase fermentation medium containingassimilable carbon, nitrogen and mineral sources, to producezearalenone.

2. The process of claim 1 wherein the microorganism is Gibberella zeae(Schw.) Petch strain 542 Keith ATCC 20273 or Gibberella zeae (Schw.)Petch strain Paul S. ATCC 20271.

3. The process of claim 2 wherein the carbon source is glucose.

4. The process of claim 3 wherein the glucose is employed in an amountof about 20 to 40 grams per 100 cc. of the fermentation medium.

5. The process of claim 2 wherein the nitrogen source is urea.

6. The process of claim 4 wherein the nitrogen source is urea which isemployed in an amount of about 0.2 to 0.8 grams per 100 cc. of thefermentation medium.

7. The process of claim 6 wherein air is conducted into the medium at arate of about 0.25 to 2 volumes, calculated at about atmosphericpressure, per volume of medium, per minute.

8. The process of claim 1 wherein the medium is maintained at atemperature of about 20 to 28 C.

9. The process of claim 6 wherein the medium is maintained at atemperature from about 20 to 28 C.

10. The process of claim 6 wherein the mineral source includes yeastextract.

7 11. The process of claim 6 wherein the fermentation mediand thenfiltered.

15. The process of claim 10 wherein the fermentation medium contains agrowth promoting amount of a hydrolyzate of protein. i

16. The process of claim 15 wherein the hydrolyzate is an enzymatichydrolyzate of casein and is present in the medium in an amount of atleast about 0.1 gram per l00 cc. of the medium.

17. The process of claim 6 wherein the fermentation medium contains aneffective amount of an osmotic pressureenhancing salt.

18. The process of claim 17 wherein the salt is selected from the groupconsisting of sodium acetate, sodium citrate, sodium succinate, sodiumchloride, and potassium chloride.

2. The process of claim 1 wherein the microorganism is Gibberella zeae(Schw.) Petch strain 542 Keith ATCC 20273 or Gibberella zeae (Schw.)Petch strain Paul S. ATCC
 20271. 3. The process of claim 2 wherein thecarbon source is glucose.
 4. The process of claim 3 wherein the glucoseis employed in an amount of about 20 to 40 grams per 100 cc. of thefermentation medium.
 5. The process of claim 2 wherein the nitrogensource is urea.
 6. The process of claim 4 wherein the nitrogen source isurEa which is employed in an amount of about 0.2 to 0.8 grams per 100cc. of the fermentation medium.
 7. The process of claim 6 wherein air isconducted into the medium at a rate of about 0.25 to 2 volumes,calculated at about atmospheric pressure, per volume of medium, perminute.
 8. The process of claim 1 wherein the medium is maintained at atemperature of about 20* to 28* C.
 9. The process of claim 6 wherein themedium is maintained at a temperature from about 20* to 28* C.
 10. Theprocess of claim 6 wherein the mineral source includes yeast extract.11. The process of claim 6 wherein the fermentation medium contains aneffective amount of a foam inhibitor.
 12. The process of claim 11wherein the foam inhibitor is a silicone oil.
 13. The process of claim10 wherein the mineral source includes phosphorus, potassium, sulfur,iron and magnesium.
 14. The process of claim 10 wherein the water in themedium is selected from the group consisting of deionized water,distilled water, and tap water which has been heated to boiling and thenfiltered.
 15. The process of claim 10 wherein the fermentation mediumcontains a growth promoting amount of a hydrolyzate of protein.
 16. Theprocess of claim 15 wherein the hydrolyzate is an enzymatic hydrolyzateof casein and is present in the medium in an amount of at least about0.1 gram per 100 cc. of the medium.
 17. The process of claim 6 whereinthe fermentation medium contains an effective amount of an osmoticpressure-enhancing salt.
 18. The process of claim 17 wherein the salt isselected from the group consisting of sodium acetate, sodium citrate,sodium succinate, sodium chloride, and potassium chloride.